Microsporidia are increasingly described as causing opportunistic infections in persons with AIDS. These parasites are difficult to diagnose because they are quite small and stain poorly with H&E. In previous studies, a histochemical diagnostics paradigm was established for screening fluids (urine, stool, mucus) with the chitin staining fluorochromes (eg. Calcofluor White), followed by corroboration with the modified trichrome stain. Diagnosis in tissues is typically made using Gram stain, or silver stain. The major disadvantages of these methods are that they are not species specific and they could be more sensitive. Polymerase chain reaction (PCR) methods therefore, were developed and utilized for detecting microsporidia. Pan microsporidian, genus specific, and species specific primers were used for PCR amplification of the small subunit rRNA gene sequences of microsporidian species known to infect humans and other mammals from formalin fixed tissues, paraffin embedded tissue sections, and formalin fixed fluids (stool, urine, mucus). The amplified products were then subjected to restriction endonuclease digestion or heteroduplex mobility shift analysis to identify the microsporidian species. If no PCR signal could be discerned, Southern analysis using species specific oligonucleotide probes was used to identify the microsporidian. Specific results include diagnosis of, a) Encephalitozoon intestinalis in human specimens (paraffin embedded kidney and brain, formalin fixed urine and stool, and unfixed alveolar lavage), b) Encephalitozoon cuniculi strain III from a culture established from puppy and human urine specimens and from human paraffin embedded kidney sections, c) Encephalitozoon hellem from formalin fixed parakeet intestine and human conjunctival scraping, and d) Enterocytozoon bieneusi in formalin fixed stools and intestinal biopsies. As these PCR based diagnostic methods are optimized for sensitivity and reliability, epidemiological studies will be performed using these methods.